This years annual report incorporates both the work of the Cell Biology Section in bone marrow failure diseases and also of the Virus Discovery Section. Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, studies have been directed towards more effective immunosuppression in AA and the application of immunosuppressive regimens to related bone marrow failure syndromes. In the clinic, studies have been directed towards more effective immunosuppression in AA and the application of immunosuppressive regimens to related bone marrow failure syndromes. In our major clinical protocol in aplastic anemia, a randomized trial comparing horse and rabbit antithymocyte globulins (ATG), nearly 100 patients have been accrued. Interim analysis of data suggested a marked and unanticipated difference in response rates at three and six months, although there is as yet no convincing difference in survival. The results of this study, which should be available publicly by the end the of the end of the year, will markedly influence the treatment of patients of this disease worldwide. Other protocols have examined the utility of a monoclonal antibody to lymphocytes, termed Campath. Campath has been employed in a variety of settings. It has produced very high hematologic response rates in patients with MDS who are selected based on a prognostic score derived from ATG treatment (pioneered in the Hematology Branch). Campath has multiple advantages compared to ATG, including greater biological uniformity, administration in an outpatient setting, and an absence of need to combine with cyclosporine and attendant toxicities. In aplastic anemia, Campath appears equivalent or better than rabbit ATG as a salvage treatment in patients who have failed horse ATG. Campath also rescues the majority of patients who relapse after horse ATG treatment. Campath is also highly effective in the therapy of large granular lymphocytic leukemia and the accompanying bone marrow failure syndrome. New studies are planned based on the results of the above trials, which will incorporate consolidation with Campath in patients treated with ATG as well additional therapies based on molecular mechanisms that underlie the bone marrow failure syndromes. In the laboratory, we have developed a method for three dimensional visualization of both mouse and human bone marrow. Necropsy or biopsy specimens are lightly fixed and then stained with a variety of fluorescent signaling markers. This method has been applied to normal tissue and also to our mouse model of aplastic anemia and murine transplant, and to patients with a variety of marrow failure and lympho- and myeloproliferative diseases. The ease of preparation of specimens and the remarkable images obtained should make this a generally interesting new method for hematologists and experimenters in hematopoiesis. Other laboratory work has focused on the importance of telomere biology to both bone marrow failure and other non-hematologic diseases. A method based on gene amplification for measurement of leukocyte telomere length, and the acquisition of sufficient numbers of healthy controls over a wide range of ages, has allowed us to apply this method to the clinic. On retrospective analysis of almost two hundred patients who have been treated at the NIH in ATG based protocols, telomere length has shown remarkable prognostic value. First, telomere length does not predict the likelihood of response to immunosuppressive therapy, suggesting that it identifies a co-factor in the development of the disease as opposed to an entirely separate syndrome, one independent of immune-mediated destruction of bone marrow. However, patients with short length have a higher rate of relapse, consistent with an anticipated lower number of hematopoietic stem cells. Most dramatically, short telomeres strongly predict malignant clonal evolution to myelodysplasia and acute myeloid leukemia. In vitro, and as predicted from murine knock-out models and from the basic science of telomere biology, we have been able to demonstrate that bone marrow from patients with short telomeres or from individuals in families in which the telomere repair genes are mutated show chromosomal instability in tissue culture, using methods such as fluorescent in situ hybridization, determination by histocchemistry of chromosomes with free telomere ends, and direct visualization of end-to-end fusion. These data provide both a practical method to predict outcomes in aplastic anemia as well as indicate a mechanism of malignant transformation based on chromosomal instability. Further, our data indicating that sex hormones can increase telomerase activity may be applicable in the clinic in the treatment of patients with bone marrow failure and short telomeres, to both improve hematopoiesis as well as to decrease the rate of malignant transformation. In separate experiments, human bone marrow cells subjected to inflammatory insults, as in mixed lymphocyte culture reactions and after exposure to toxic proteins such as gamma-interferon, also show accelerated telomere attrition, chromosome instability, and aneuploidy. Therefore, telomere biology may represent the nexus between chronic inflammation and cancer. TERT and TERC mutations, present in patients with aplastic anemia and acute myeloid leukemia, have also been identified in a total of five pedigrees in individuals with significant liver disease. Nodular regeneration appears to be a common feature of the pathology. In at least one instance, identification of a mutation explains our previously reported association of liver disease and aplastic anemia. Almost 100 patients with a variety of serious liver disease, from the University of Arizona have been similarly assessed for these mutations, which have been found in approximately 5% of unselected cases;these results suggest that telomere repair mutations represent risk factors for cirrhosis. In our virus laboratory, efforts have been concentrated on the analysis of human samples available from a phase I vaccine trial of empty capsids, generated commercially, in a baculovirus system first developed in the Hematology Branch. In this protocol, a significant proportion of patients did produce anti-B19 parvovirus IgG. Furthermore, efforts were directed at improving the efficiency of empty capsid formation by modifications of the original tissue culture system, which if successful would potentially provide patent protection and facilitate practical development of the vaccine.